The invention relates to the structural analysis of proteins in the molecular mass range from approximately 5 to 100 kilodaltons, without prior enzymatic digestion to small peptides, in mass spectrometers that operate with ion traps.
Proteins and peptides both consist of chains of amino acids; the difference between peptides and proteins consists only in the length of the chains, but there is no sharply defined distinction. Proteins consist of chains with many more than 20 linked amino acids, whereas peptides generally consist of fewer. When we speak here of proteins, chains with around 40 or more amino acids are meant, with molecular masses greater than around 5 kilodaltons. The majority of proteins have a molecular mass below 100 kilodaltons; these “medium-mass proteins” of 5 to 100 kilodaltons are the primary focus of interest here, although some other interesting proteins, such as antibodies, have molecular masses between 100 and 1,000 kilodaltons. There are even some proteins with masses of more than a megadalton. Until now, the very large proteins have scarcely been accessible to mass spectrometric top-down analysis without prior enzymatic digestion. There are, however, enzymes whose cleavages only occur at rarely found sequence patterns, and which generate large digestion proteins with molecular masses above 5 kilodaltons. These large digestion proteins will also be considered here as “medium-mass proteins”. Such enzymes also permit piece-wise top-down analysis of very large proteins.
The mass spectrometric structural analysis of heavier proteins, which essentially consists in analyzing the sequence of amino acids, but also includes the identification of their modifications, starts conventionally with enzymatic digestion of the proteins to create relatively light digestion peptides, in order to produce molecular sizes that can effectively be measured in a mass spectrometer. The most frequently applied technique of trypsin digestion, for instance, yields digestion peptides with an average length of 10 amino acids, as trypsin exclusively cleaves the C-terminal peptide bonds of the amino acids arginine and lysine. Other enzymes that only cleave the peptide bond specifically at one amino acid yield digestion peptides with an average length of twenty amino acids. The digestion peptides are then fed to a tandem mass spectrometer, either as an unseparated mixture, or following chromatographic separation. Measurement of the fragment ion spectra of the individual digestion peptides now provides partial segments from the sequence. These are usually sufficient to perform identification in protein sequence databases, using suitable search engines, and also to identify some of the modifications.
Unfortunately, this method also has disadvantages. Generally only between 50 and 70 percent of the digestion peptides can be found and measured in the mass spectrometer. No information is yielded regarding the modifications of the lost digestion peptides. The molecular mass of the undigested protein cannot be determined. The information about the sequence of digestion peptides in the original protein is lost. If the digestion acts on two or more proteins that cannot be separated, it is not possible to assign the digestion peptides to the specific proteins. For this reason methods have been sought for some time, by which the protein as a whole can be subjected to mass spectrometric analysis without prior enzymatic digestion, and which permits determination of the longest possible sequence segments from the original protein molecules. Such methods have so far only been developed for very expensive ion cyclotron resonance mass spectrometers (ICR-MS). They have become known as “top-down analyses”. Top-down analyses transfer the process of dividing the proteins into smaller units to the mass spectrometer; they require appropriate ionization and fragmentation methods for the medium-sized proteins and, as shown below, of further measures for equalizing the very complex fragment ion spectra that result.
Matrix assisted laser desorption (MALDI) and electrospraying (ESI) are predominantly used nowadays for ionizing proteins and other biomolecules. MALDI almost exclusively yields singly charged ions, which are very unfavorable for fragmentation because they are difficult to fragment, and because their fragment ions only yield small segments of the amino acid sequence, with large gaps. The complex process known as MALDI yields both a non-ergodic spontaneous fragmentation in the laser plasma (ISD=“in-source decay”) as well as an ergodic fragmentation through the metastable decay of excited analyte ions (LID=“laser induced decomposition”, also known as PSD=“post-source decay”), but both can only be used for measurement in highly specialized MALDI time-of-flight mass spectrometers, and will not be considered further here.
If multiply charged analyte ions are to be generated for mass spectrometric analysis of biomolecules, in particular for an analysis that includes fragmentation, the usual method of generating molecular ions is electrospray ionization (ESI), which creates ions at atmospheric pressure outside the mass spectrometer. These ions are then guided through inlet systems of a known type into the mass spectrometer's vacuum system, and on into mass spectrometric analyzer, e.g., an ion trap. There the molecules are accessible to further manipulations such as fragmentation.
Electrospray ionization produces almost no fragment ions, and the ions are substantially those of the protonated molecules; due to their multiple protonation their mass is a corresponding number of Daltons greater than the neutral molecules, and they are therefore frequently referred to as “pseudomolecular ions”. Multiple protonation from electrospraying usually results in multiply charged ions of the molecules: doubly and triply charged ions for smaller molecules such as peptides, while for larger biomolecules, such as proteins with molecular masses in the range between 5 and 100 kilodaltons, the ions may carry up to ten or even a hundred charges. If possible, fragmentation is carried out on protein ions with double to quadruple charges, as these have a very high yield of fragment ions and deliver easily evaluated fragment ion spectra. In the case of medium-sized proteins, however, molecular ions with two to four charges only occur with vanishingly small intensities, and cannot therefore be used for fragmentation.
The spectra of the fragment ions are also known as “daughter ion spectra” of the parent ions concerned. It is also possible to measure the “granddaughter spectra”, which are the fragment ion spectra of selected daughter ions. The structures of the fragmented ions can be read from these daughter (and granddaughter) ion spectra; for instance, it is possible (although difficult for many fragmentation methods) to determine at least parts of the sequence of amino acids in a peptide or protein from these spectra.
Two different types of ion traps exist nowadays, frequently referred to as “Penning ion traps” and “Paul ion traps”. The Penning ion traps hold the ions radially in a strong magnetic field, and axially in an electric trapping potential. They are used in ion cyclotron resonance mass spectrometers (ICR-MS). Modern ICR-MS nearly all use very expensive superconducting magnet coils, cooled in liquid helium, to generate magnetic fields with very high strengths of around 7 to 15 Tesla. Fewer than one thousand such instruments have been built. Nowadays they are predominantly also equipped with RF ion traps to permit collision-induced dissociation, or other manipulations of the ions.
Paul ion traps hold the ions using inhomogeneous RF fields. These create what are known as “pseudopotentials”, which form a storage well in which both positive and negative ions can be trapped. In “three-dimensional” (3D) RF ion traps, the pseudopotential rises in all three spatial directions, whereas they rise only in two spatial directions in “two-dimensional” (2D) RF ion traps. In two-dimensional ion traps, the ions must be held in the third spatial direction by other techniques, usually by DC potentials. In the potential wells of the RF ion traps, the ions can carry out what is referred to as “secular oscillations”. The oscillation frequency is inversely proportional to their mass-to-charge ratio m/z. Filling the trap with a collision gas damps the secular oscillations, and the ions accumulate as a cloud at the minimum of the potential well. The RF ion traps are very cheap in relation to their performance, and have therefore become extremely widespread, with many thousands of instruments in use. As will be explained in more detail below, Paul ion traps can be designed as what are known as 2D ion traps or as 3D ion traps.
Mass spectrometers with RF ion traps have features that make them interesting for many types of analysis. In particular, they can isolate selected types of ions (the parent ions) in the ion trap and fragment them using a variety of methods. The isolation of one type of ion means that all the uninteresting ion types are removed from the ion trap by strong resonant excitation of their secular oscillations or by other measures, so that only the parent ions remain. These can then be fragmented, yielding fragment ion spectra uncontaminated by fragment ions of other substances.
RF ion traps have one special feature that can sometimes be disadvantageous. They possess a “lower mass threshold” for ion storage. Ions whose mass-to-charge ratio m/z is below this mass threshold cannot be stored in the ion trap. These light ions can be accelerated so much in a single half-wave of the RF voltage that they collide with the electrodes and are destroyed. The lower mass threshold rises in proportion to the RF voltage.
Nowadays, two fundamentally different types of fragmentation are available in the different types of ion traps: “ergodic” fragmentation and “electron-induced” fragmentation.
“Ergodic” fragmentation of analyte ions refers here to a fragmentation in which a sufficiently large excess of internal energy in the analyte ions leads to fragmentation. This excess of inner energy can, for instance, be generated by a large number of collisions between the analyte ions and a collision gas, or by the absorption of a large number of photons from infrared radiation.
According to the “ergodic hypothesis” originally formulated by Boltzmann, in a closed system such as that of a complex molecular analyte ion, when sufficient energy is present, then every state that can be achieved with this energy will in fact be achieved over the course of time. This ergodic hypothesis has since been proved mathematically, and is therefore no longer strictly a hypothesis. Since the fragmentation represents a possible state, that is the generation of two smaller particles from the analyte ion, the fragmentation will occur at some time. The absorption of energy temporarily creates “metastable” analyte ions, which then at some point in time decompose. The decomposition itself is characterized by a “half life time” which, however, depends on the quantity of excess inner energy and cannot be quantified because the amount of excess energy is unknown.
The probability that a given bond will experience an ergodic cleavage depends on its binding energy. The weakest bonds in the analyte ion have the highest probability to be cleaved. In proteins, the weakest bonds are those known as “peptide bonds” between the amino acids, leading to fragments in the so called b and y series, which occur partly as fragment ions and partly as neutral particles. Since the different peptide bonds between different amino acids have somewhat different binding energies, some peptide bonds in the analyte ion are cleaved with a greater probability and others with a lower probability. As a result, not all the fragment ions created by cleaving peptide bonds have the same intensity in the fragment ion spectrum. Non-peptide bonds within the chain of amino acids are cleaved so rarely that the resulting particles are not found in measurable quantities. Modifying side chains, however, like phosphorylations or glycosylations, are regularly split off.
The conventional method of fragmenting analyte ions in RF ion traps is ergodic fragmentation through collisions, in which the excess of internal energy is introduced to the analyte ions through numerous collisions with a collision gas in the RF ion trap. To enable the collisions to pump energy into the analyte ions, they must occur with a certain minimum collision energy. The collision energy is conventionally created by weak, resonant excitation of the secular ion oscillations of the parent ions by means of a dipolar alternating voltage. This leads to a large number of collisions with the collision gas without removing the ions from the RF ion trap. The ions can accumulate energy through these collisions, finally resulting in ergodic decomposition of the ions and the creation of fragment ions. Until a few years ago, this collision-induced dissociation (CID) was the only known method of fragmentation in RF ion traps.
Collision-induced dissociation in RF ion traps also has disadvantages, however. For larger analyte ions, for example, it is necessary to use a very high RF voltage for storing the ions in order to create sufficiently strong collision conditions. As a result, the RF ion trap has a very high lower mass threshold. Ions with a mass below the mass threshold can no longer be stored, and are lost. The fragment ion spectrum therefore only begins at a mass that is around one third of the mass m/z of the analyte ion; the fragment ion spectrum no longer yields any information about the light fragment ions, as those ions have been lost. Multiply charged heavy analyte ions often have a low mass-to-charge ratio m/z of only around 500 to 1,000 Daltons because of the large number of protons; these analyte ions cannot be fragmented at all, as the RF voltage cannot be set high enough to generate sufficiently energetic collisions to pump energy into the ions.
To also store very small fragment ions (in particular what are known as the immonium ions, created by the internal fragmentation of fragment ions) by means of collision-induced dissociation, special methods have recently become known that make use of the slow, metastable decay of the ions by the ergodic fragmentation process. The method is described in patent DE 10 2005 025 497 B4 (A. Brekenfeld; equivalent to patent application publication GB 2 428 515 A).
Collision-induced dissociation in ion cyclotron resonance mass spectrometers (ICR-MS) is very difficult, as these spectrometers only work well at optimal ultra-high vacuums below 10-9 hectopascal. Nevertheless, equipment for collision-induced dissociation is available for the ion traps of these devices. An other kind of ergodic fragmentation, however, was introduced here at a very early stage: infrared multiphoton dissociation (IRMPD). With this method, the internal energy of the analyte ions is increased through the absorption of a large number of infrared photons. Carbon dioxide lasers are usually used here to generate sufficiently strong infrared radiation. IRMPD equipment for ICR-MS is commercially manufactured and marketed.
Document WO 02/101 787 A1 (S. A. Hofstadler and J. J. Drader) has published that infrared multiphoton dissociation (IRMPD) can also be used in RF ion traps. The infrared radiation is introduced into a three-dimensional RF ion trap in a simple manner through an evacuated hollow fiber with an optically reflective internal coating. This makes a further process for ergodic fragmentation available in RF ion traps. This type of fragmentation is very favorable, as it can be carried out at low RF voltages; the small fragment ions are then also stored. However, there are not yet any commercially marketed RF ion trap mass spectrometers featuring this method of fragmentation.
We turn now to electron-induced fragmentation methods. About ten years ago, an entirely new method of fragmenting protein ions was discovered: non-ergodic fragmentation, induced by the capture of low-energy electrons (ECD=“electron capture dissociation”). Through the direct neutralization of an associated proton, which is then lost as a radical hydrogen atom, the potential balance of the protein ion is disturbed in such a way that appropriate repositionings induce a cleavage of the amino acid chain. The cleavage does not occur at the peptide bonds, but at neighboring bonds, leading to what are known as c and z fragment ions.
It is particularly easy to carry out this type of fragmentation in ICR mass spectrometers, as the low-energy electrons from a thermionic cathode can simply be guided along the magnetic force lines to the stored cloud of analyte ions. ECD fragmentation can only be applied to RF ion traps with some difficulty, as the strong RF fields do not efficiently allow the electrons to reach the cloud of analyte ions with low-energy. There are, nevertheless, a variety of approaches to ECD fragmentation in RF ion traps, but each of them requires more costly equipment.
Recently, a method was published for the fragmentation of ions in RF ion traps that delivers the same kind of fragmentations as electron capture dissociation (ECD) but by means of different reactions: electron transfer dissociation (ETD). This can be easily done in ion traps by introducing suitable negative ions in addition to the stored analyte ions. Methods of this type have been described in the published patent applications DE 10 2005 004 324.0 (R. Hartmer and A. Brekenfeld) and US 2005/0199804 A1 (D. F. Hunt et al.). The fragment ions here (as in the case of ECD) belong to what are known as the c and z series, and are therefore very different from the fragment ions of the b and y series, which are obtained by ergodic fragmentation. The fragments in the c and z series have significant advantages for determining the amino acid sequence from the mass-spectrometric data, not least because ETD fragment ion spectra can more easily extend down to smaller masses than collision-induced fragment ion spectra.
The fragmentation of protein ions by electron transfer (ETD) in an RF ion trap is created in a very simple manner by reactions between multiply charged positive protein ions and suitable negative ions. Suitable negative ions are often radical anions, such as those of fluoranthene, fluorenone, anthracene or other polyaromatic compounds. In radical anions, the chemical valences are not saturated, which permits the easy donation of electrons in order to reach an energetically favorable non-radical form. They are generated in NCI (negative chemical ionization) ion sources, most probably by simple electron capture or by electron transfer. In principle the design of NCI ion sources is the same as for chemical ionization (Cl ion sources), but they are operated in a different way in order to obtain large quantities of low-energy electrons. NCI ion sources are also referred to as electron attachment ion sources.
It has since become known that electron transfer can take place from highly excited neutral particles, for example by highly excited helium atoms from a “fast atom bombardment” (FAB) particle source (DE 10 2005 005 743 A1, R. Zubarev et al.). This type of fragmentation is abbreviated to MAID (“metastable atom induced dissociation”). Here again, the fragment ion spectra are similar to those obtained from ECD. The source of the electron appears to be irrelevant for the non-ergodic fragmentation process by neutralization of a proton by an electron. The ECD, ETD and MAID fragmentation methods can therefore all be referred to as “electron induced” fragmentation methods.
It is very easy to evaluate the fragment ion spectra if they are produced from parent ions with between two and about four charges, because fragment ions with between two and four charges can be recognized as such from the differences in mass of their isotope pattern, and because the fragment ion spectra are not too complex. It is a different situation when highly charged parent ions having, for instance, ten or thirty charges, are subjected to this fragmentation procedure. The number of different types of fragment ion is extremely high and the great majority of fragment ions are crowded in a region around the mass-to-charge ratio m/z of about 600 to 1,200 Daltons. The fragment ion spectrum is so complex that it is scarcely possible to evaluate it, particularly as the isotope patterns in the RF ion traps used as mass analyzers can no longer be resolved by mass, and therefore the level of charge cannot be established.
Larger molecules, proteins in particular, yield multiply charged ions in electrospray ion sources; as a rule of thumb, we can assume that every increase in mass by 800 to 1,000 Daltons leads to a mean increase in charge of one proton. A protein with a mass of 10,000 Daltons has therefore accepted about 10 to 12 protons at the peak of the charge distribution, but in most cases there is a broad distribution of ions with various numbers of charges, most of them being in the mass-to-charge ratio m/z range from 600 to 1,200 Daltons. Doubly or triply charged ions here occur with vanishingly small frequencies, and therefore cannot practicably be used for generating the fragment ions; for these reasons fragmentation comes up against great difficulties with protein molecules in the range of molecular masses between 5 and 100 kilodaltons, even though the highly charged analyte ions can be dissociated extremely well by electron transfer, for instance. The fragment ions created this way, in particular the heavy fragment ions, are themselves predominantly highly charged, and form the complex fragment ion spectrum described above.
It has long been known that ions with multiple charges can be converted by continued deprotonation (“charge stripping”) into ions with single or low numbers of charges. This is done very easily by continued proton transfer from the ions with multiple positive charges to special kinds of negatively charged ions, most particularly to non-radical anions, which are thereby neutralized. The reaction cross-section for such a proton transfer reaction is proportional to the square of the number of proton charges on the positively charged ion; the deprotonation therefore happens very quickly for highly charged ions, while the reaction speed is sharply reduced when the ions have lower charges. If, for instance, the supply of negative reactant ions for deprotonation is stopped when singly charged ions are reached, the measurements in the mass analyzer will yield mass spectra which can easily be evaluated, as these now contain practically only the signals of singly charged ions.
For electron transfer dissociation of medium-sized proteins in ion traps, it is already known that this effect can also be applied to the fragment ions created in this way: after highly charged ions of the large protein molecules have been stored, ETD fragmentation is applied by injecting suitable radical anions; then, non-radical anions are injected for deprotonation, until almost complete reduction of the charge states down to singly charged ions has occurred. This yields mass spectra of the ETD fragment ions which can easily be evaluated. A report was presented to the “17th International Mass Spectrometry Conference”, Aug. 27-Sep. 1, 2006, Prague, by Donald F. Hunt (Abstract 1.2).
A further interesting method has recently become known in the field of deprotonation of highly charged pseudomolecular ions. The highly charged pseudomolecular ions of a substance that are present with various levels of charge can be deprotonated simultaneously in an RF ion trap, and the process of deprotonation can be halted at a particular level of charge so that all the pseudomolecular ions from higher levels of charge accumulate at this particular charge level in a partially deprotonated state. To do this, it is necessary to generate gentle resonant excitation of the secular oscillations—by means of a dipolar alternating voltage—at the mass-to-charge m/z of this charge level of the pseudomolecular ions. The ions that then are in forced oscillation at this charge level are no longer able to participate in further reactions with deprotonating reactant anions, as deprotonation requires a low relative velocity of the participating particles. This method is described in U.S. Pat. No. 7,064,317 B2 (S. M. McLucky et al.).
Such a conversion of highly charged pseudomolecular ions of various charge levels to a specified level of charge brings, at the same time, a high sensitivity, as the analyte ions of all the higher charge levels accumulate with a relatively high yield at the chosen level during the deprotonation process. Yields of more than 50 percent can be achieved. Furthermore, if highly charged ions of several substances are present, it is thus possible to select only the analyte ions, as the ions of the other substances are not collected, but, if the reaction time is long enough, undergo deprotonation to the bitter end, that is until they are neutralized.
It was already explained above that an extraordinary number of fragments are formed from medium-mass, highly charged protein ions, and these also carry a wide range of numbers of charges. The majority of fragment ions appear in the mass-to-charge ratio m/z range from 600 to 1,200 Daltons. The resulting mass spectrum is, in most cases, impossible to untangle, even with the highest possible mass resolution. So many fragment ions, each with its isotope pattern, are superimposed that even the best deconvolution algorithms are not able to cope with this mixture of signals. If the RF ion traps of the mass spectrometers are also being used as mass analyzers, the position is hopeless, as they are not capable of resolving the isotope patterns of the highly charged fragment ions into the individual masses.
If, however, as is known from the prior art, the fragment ions in the ion trap are deprotonated down to a charge level of one (or at most two), it becomes possible to measure a mass spectrum for the fragment ions. This can in fact be done both with RF ion traps that are also used as mass analyzers and with other types of mass analyzers. A disadvantage, however, is that the mass spectrum is restricted to the mass analyzer's mass range for singly (or at most doubly) charged ions. This does not give good sequence coverage for medium-mass proteins.
The measurement of a mass spectrum for the fragment ions can be done with the RF ion trap itself. A range of scan methods are known for this, almost all of which are based on a rapid sequence of mass-selective ion ejections. The ejected ions are measured in an ion detector. The fragment ions can, however, also be measured in connected mass analyzers of another type. For example, combinations of RF ion traps with ion cyclotron resonance mass spectrometers, with Kingdon cells or with time-of-flight mass spectrometers are commercially available.
Three-dimensional Paul RF ion traps (3D ion traps) consist of a ring electrode and two end cap electrodes. As a general rule, the RF voltage is applied to the ring electrode, but other operating modes are possible. Both positive and negative ions can be held in the quadrupole RF field inside the ion trap for mass spectrometric analysis. The ion traps can be used as mass spectrometers by ejecting the stored ions—selected according to mass—and measuring them with secondary electron multipliers. Several different methods are known for the ion ejection, but these will not be described in any further detail here. Good commercial ion trap mass spectrometers have a mass range extending up to a mass-to-charge ratio of m/z=3,000 Daltons, and at each mass it is possible with special scan methods to resolve the isotope pattern up to ions with four charges. Ion trap mass spectrometers are amongst the cheapest mass spectrometers, and are widely used.
Linear RF ion traps (also known as 2D ion traps because the electrical fields in the interior only change in two dimensions) consist of two or more pairs of pole rods supplied with RF voltage, and end electrodes whose inhomogeneous RF potentials can repel both positive and negative ions. If it is desired to store both positive and negative ions simultaneously, special steps must be taken with respect to their storage in the axial direction. For instance, RF voltages can be used to generate pseudopotentials at the ends in order to retain ions of both polarities. Two-dimensional ion traps with four pole rods form an internal quadrupole field, and can be used as mass analyzers in the same way as 3D ion traps. Here again there are different scanning procedures, such as those using the mass-selective ejection of ions through slots in the pole rods, or through diaphragms at the end of the rod systems. Commercial devices of this type at present cover a range of mass-to-charge ratios extending up to m/z=2,000 Daltons.
It is a particular feature of all RF ion trap mass spectrometers, that the absolute mass resolution Ra=1/Δm is constant, and not the relative mass resolution Rr=m/Δm as with other types of mass spectrometer. This means that the width of the ion signals in ion trap mass spectrometers is constant over the whole mass range (measured in terms of the mass-to-charge ratio, m/z), whereas for practically every other kind of mass spectrometer, the width of the ion signals grows in proportion to the mass-to-charge ratio m/z. For ion trap mass spectrometers, therefore, the resolution for an isotope pattern increases under deprotonation; for all other types of mass spectrometer, the resolution for the isotope pattern is approximately constant under deprotonation.
Internally, quadrupole ion traps contain a principally quadrupole RF electrical field that drives ions above the lower mass threshold toward the center, as a result of which the ions in this field are subject to what are called secular oscillations. The restoring forces in the ion trap can be described by what is referred to as a pseudopotential, given by a temporal averaging of the forces acting on an ion in forced oscillation in the real potential. The pseudopotential rises quadratically in two or three spatial directions. The ions, both positive and negative, can oscillate in this pseudopotential “well”.
The presence of a collision gas in the ion trap has the effect of decelerating the original oscillations (the secular oscillations) of the ions in the pseudopotential well; the ions then collect as a small cloud in the center of the ion trap. In usual ion traps, typically filled with some tens of thousands of ions, the diameter of the cloud is around one millimeter. In 3D ion traps the cloud is elliptical in shape, whereas in 2D ion traps it takes the form of an elongated thread. The diameter is determined by an equilibrium between the restoring force of the pseudopotential and the repulsive Coulomb force between the ions. Residual thermal energies enlarge the ion cloud by a very small amount.
RF ion trap mass spectrometers equipped with special ion sources for the production of negatively charged reactant ions are available commercially. They can be used to create both radical anions for fragmentation by electron transfer and non-radical anions for reducing the number of protons of analyte ions by the transfer of protons from the analyte ions to the negative reactant ions. Negative ions for deprotonation can, however, also be made in the electrospray ion sources with which the vast majority of ion trap mass spectrometers are equipped.
It is particularly favorable for de novo sequencing, and also for spectral evaluation purposes, to record ergodic fragment ion spectra along with electron-induced fragment ion spectra. De novo sequencing is always desirable when a search engine fails to find any reasonable results in a protein sequence database because, for instance, a protein of this type is not yet present in the database. A comparison of the ergodic and electron-induced fragment ion spectra allows the ion signals to be immediately assigned to the c/b series or the z/y series. This is because there are fixed mass differences between the c-ions and the b-ions, as there are between the z-ions and the y-ions, from which the association can easily be seen. As a result, partial sequences for both series of fragment ions can easily be read. Modifications are also easy to identify, as side chains like phosphorylations or glycosylations are preserved with electron-induced fragmentation, whereas they are lost in ergodic fragmentation. The differences make the modifications visible.
The easy generation of ETD fragment ion spectra therefore does not mean that the generation of ergodic fragment ion spectra is superfluous, as a great deal of valuable information is only obtained by putting the two types of fragment ion spectra side-by-side.